Vectors, transformed cells and process for the preparation of hirudin

ABSTRACT

The present invention relates to vectors for cloning, in a host cell, hirudin or an analog of hirudin, characterized in that it comprises the gene for encoding hirudin or an analog of hirudin, and the elements for the expression of this gene in said host cell, said coding gene beginning, after the starting sequence, by an Ile codon and a Thr codon. This invention also relates to the process for the preparation of hirudin or analogs of hirudin as well as the hirudin or anologs of hirudin obtained.

This application is a continuation, of application Ser. No. 08/312,972,filed Sep. 30, 1994, which is a continuation of application Ser. No.08/159,697, filed Dec. 1, 1993, which is a continuation of applicationSer. No. 08/026,220, filed Mar. 1, 1993; which is a continuation ofapplication Ser. No. 07/793,287, filed Nov. 13, 1991; which is acontinuation of application Ser. No. 07/554,076, filed Jul. 16, 1990;which is a continuation of application Ser. No. 07/432,318, filed Nov.3, 1989; which is a continuation of application Ser. No. 06/808,447,filed Nov. 25, 1985, all now abandoned.

The present invention relates to vectors for the cloning and expressionof the DNA sequence coding for hirudin or hirudin analogs,microorganisms transformed by these vectors and processes which enablehirudin to be obtained by fermentation, as well as the hirudin obtained.

The anticoagulant activity present in the salivary glands of themedicinal leeches Hirudo medicinalis originates from a small polypeptideknown as hirudin. This very specific and very effective thrombininhibitor has been widely studied recently, since it potentiallyrepresents a very useful therapeutic agent. However, the extremedifficulty and cost in isolating and purifying it has prevented it frombeing used more widely, or even from being studied at the clinicallevel. The cost of the material is on the order of 1,300 to 1,600 francs(1983) for 2,000 U.

The present invention relates to a means for producing hirudin bycloning the genes and the expression thereof by means of the recombinantDNA technique in a heterologous host cell, in order to obtain a largeamount of polypeptide having the biological properties of hirudin.

The polypeptide with anti-thrombin activity obtained from the salivaryglands of leeches was isolated for the first time in the middle of 1950(1, 2). This protein, known as hirudin, was purified approximately 650times, starting from the heads of leeches, to obtain a specific activityof 8,500 units per milligram.

The molecular weight of the protein was then estimated at approximately16,000. This protein was stable to denaturation by heat and had anisoelectric point of 4.8. In electrophoresis on paper, the purifiedproduct migrates in the form of a single band and amino acid analysisshows that the material is very rich in acidic amino acids, asparticacid and glutamic acid.

Further stages of purification (3) increase the specific activity to10,400 U/mg, and isoleucine was identified as the N-terminal amino acid(3, 4).

It was possible to demonstrate that hirudin activity resisted digestionwith the proteolytic enzymes plasmin, chymotrypsin A and trypsin, butwas sensitive to digestion with papain, pepsin and subtilopeptidase A(3).

The estimated molecular weight is now slightly less than it was in theinitial papers, and this molecular weight is regarded as being of theorder of 10,000 daltons. The first estimate of the dissociation constantof the 1:1 thrombin-hirudin complex (0.8×10⁻¹⁰) indicates an extremelystrong association between these two molecules (5). For practicalreasons, the non-covalent complex between these two molecules can beregarded as non-dissociable in vivo.

The mechanism of action of hirudin as an anticoagulant is only justbeginning to be understood (5). The substrate for the binding of hirudinis thrombin, which is a proteolytic enzyme which, through activation (bymeans of the activated factor X) from its zymogen form, prothrombin,cleaves the fibrinogen in the circulatory stream to convert it tofibrin, which is required for the formation of the blood clot.

Hirudin is a very specific thrombin inhibitor, which reacts more rapidlywith thrombin than fibrinogen does. Furthermore, it is not necessary tohave other clotting factors or other plasma constituents present. Theinteraction between the two molecules is at least partially due to theirionic interaction, since acetylation of the free amino groups ofthrombin produces a loss in the binding of hirudin. Furthermore, hirudinbinds in the same sites as those occupied by the fibrinopeptide and noton the active sites of the thrombin, since acetylated thrombin retainsits esterase activity which is not inhibited by hirudin, and sincethrombin treated with DFP (diisopropyl fluorophosphate), whichphosphorylates the serine-active centers in thrombin, continues to bindhirudin.

The next development in the study of hirudin consisted of thedevelopment of a process for extracting hirudin from whole leeches (16)instead of the very awkward process which consisted in dissecting thehead of the animal. The final product obtained by this process has abiological activity similar to that of the hirudin obtained from theheads, but has a specific activity of 6,500 antithrombin units permilligram.

The estimated size of this compound, determined by equilibriumsedimentation, is 12,000, but the important difference between thispreparation and the preceding preparations is that valine was identifiedas the N-terminal amino acid instead of isoleucine, which had originallybeen found. The cause of this difference was apparently explained whenit was demonstrated that the second component of the N-terminal endturned out to be valine, and since the dansyl dipeptide val--val isresistant to acid hydrolysis, it was at first thought (7) that theinitial N-terminal end with a val-val dipeptide had been confused withthe isoleucine derivative, since these components are not well resolvedby the chromatographic separation used.

A preparation of hirudin from the whole animal was used to determine theamino acid sequence of the protein (8, 9) which is shown in FIG. 1.There appears to be no carbohydrate attached to hirudin, but thetyrosine residue at position 63 is modified by an O-sulfate ester group.

The function of this modification is unknown, but it is significant thata similar modification also occurs in the fibrinopeptide B of a largenumber of animal species (9).

A recent study (10) has demonstrated that, when the sulfate ester ismade to disappear completely, the activity is reduced to only 55% of theinitial hirudin activity.

The question regarding the nature of the N-terminal end of hirudin hascoming up again in studies (12, 13) which appear to indicate that thereare two different forms of hirudin; one form having low activity, knownas pseudo-hirudin, which is thought to have been extracted from thebodies of leeches, with the val--val sequence at the N-terminal end; anda form which predominates in the heads, which appears to be highlyactive and to have an isoleucine radical at its N-terminal end.

The specific and very substantial antithrombin activity of hirudinimmediately suggests a clinical application, that is to say, itsapplication as an anticoagulant.

Hirudin has been the subject of much study in animals for itsanticoagulant properties. The most detailed study (14) describes theactivity of hirudin in the prevention of venous thromboses, vascularocclusions and disseminated intravascular coagulations (DIC) in rats.Hirudin is well tolerated by rats, dogs, rabbits and mice when it is ina highly purified form and is injected intravenously. The LD₅₀ in miceis greater, 500,000 U/kg of body weight (that is to say, 60 mg/kg).Another study (15) shows that mice tolerate doses ranging up to 1 g perkg and that rabbits tolerate up to 10 mg/kg both intravenously andsubcutaneously. In mice, repeated injections over a period of two weeksdo not lead to sensitization reactions. Two other independent studies,one using dogs (16) and the other (17) demonstrating the activity ofhirudin in the prevention of DIC in rats, concur with the positiveresults of Markwardt and his co-workers.

It has also been possible to demonstrate that hirudin counteracts theendotoxins induced by DIC in pigs, and thus constitutes a potentialsolution to the very serious problems caused by endotoxinemias whichlead to high mortality in pigs. Furthermore, hirudin in experimentalanimals is rapidly eliminated (it has a half-life on the order of 1hour) in a still biologically active form, by way of the kidneys.

This study suggests that hirudin can constitute a useful clinical agentas an anticoagulant. Furthermore, the pre-phase of blood coagulation isnot affected in view of the high specificity of the action of hirudin.The antithrombin activity is dependent on the dose, and the effect ofhirudin is rapidly reversible in view of its rapid renal elimination. Ithas been possible to demonstrate that hirudin is far superior to heparinfor treating DIC (14, 17), as could be expected in view of the fact thatDIC is accompanied by a decrease in antithrombin III (a cofactorrequired for the action of heparin) and a salting-out of platelet factor4, which is a very effective anti-heparin agent.

One of the studies has demonstrated the possibility that hirudin may beabsorbed by the skin of humans (19), although the results obtainedremain somewhat difficult to interpret.

Commercial preparations of crude leech extracts are available(Hirucreme, Exhirud-Blutgel), but further tests with larger doses of ahighly purified material are needed to establish whether this is auseful administration route.

In general, the preferred administration routes are the intravenous,intramuscular and percutaneous routes. Other administration routes havebeen reported for hirudin, in particular, the oral route (BSM 3,792M).

In combination with other components, this product can also be used inthe treatment of psoriasis and other cutaneous disorders of the sametype, as described in DOS 2,101,393.

Hirudin can, in addition, be used as an anticoagulant in clinical testsin the laboratory, and a research tool. In this case, the highspecificity for a single stage in the coagulation of the blood can havea substantial advantage over the anticoagulants which are commonly usedand which are much less specific in their action.

Furthermore, hirudin can be very useful as an anticoagulant agent inextracorporeal circuits and in dialysis systems, where it can havesubstantial advantages over other anticoagulants, especially if it canbe immobilized in an active form on the surface of these artificialcirculatory systems.

Finally, the use of labeled hirudin can constitute a simple andeffective method for measuring the levels of thrombin and prothrombin.

In summary, hirudin has a large number of possible applications:

1) as an anticoagulant in critical thrombotic conditions, for theprophylaxis and prevention of extension of the existing thromboses;

2) as an anticoagulant for reducing hematomas and swellings aftermicrosurgery, since substantial use is made of living leeches;

3) as an anticoagulant in extracorporeal circulation systems and as ananticoagulant agent for coating synthetic biomaterials;

4) as an anticoagulant in clinical tests on blood samples in laboratoryexperiments;

5) as an anticoagulant in clinical research on coagulation and as anexperimental tool;

6) as a possible topical agent for cutaneous application in thetreatment of hemorrhoids, varicose veins and edema; and

7) as a component in the treatment of psoriasis and other relateddisorders.

Finally, hirudin can be used to bind thrombin in media in which thrombincauses interference (an assay, an experiment or treated blood, forexample). In particular, hirudin permits coagulation to be limited inextracorporeal circuits.

Labeled hirudin can, in addition, be used to detect clot formation. Ineffect, clot formation demands the conversion of circulating prothrombinto thrombin, to which the hirudin becomes selectively bound. Thedetection of an accumulation of labeled hirudin at a point in thepatient's body permits the formation of a clot to be visualized.

Despite these many advantages as an anticoagulant, hirudin has nothitherto been used, widely even in clinical research. This is due to thefact that the natural material is very difficult to obtain in pure form,and above all, that it is particularly expensive to even begin clinicaltrials in order to demonstrate a potential use. Although there areadequate purification processes (20, 21) for obtaining very puresamples, the difficulty of obtaining the basic material (leeches) insufficient amount remains the major obstacle.

Although hirudin is sold commercially by various companies (Sigma,Plantorgan, Pentopharm), such preparations show an activity which canvary enormously, and are highly variable in their purity.

For this reason, the production of hirudin by recombinant DNA technologyis an especially attractive solution for obtaining this material inlarge quantities and at reasonable cost, in order to permit this type ofproduct to be tested and used.

In the discussion which follows, the term "hirudin" will, for the mostpart, be used. However, it will be understood from the above and fromadditional factors which emerged from the present study, that there areseveral forms of hirudin, and consequently, the term "a hirudin" wouldbe the more correct term.

For this reason, in the discussion which follows, it will be understoodthat the term "hirudin" refers to any one of the forms of natural orsynthetic hirudin, that is to say, a product having the same activity invivo as hirudin, which will sometimes be referred to as a "hirudinanalog".

It is, moreover, appropriate to note that the products referred to as"hirudin analogs", obtained from bacteria, are devoid of the O-sulfateester function but that, on the other hand, "bacterial hirudin maycontain at the N-terminal end a methionine amino acid which does notappear in the native hirudin. But it is clear that, the term "hirudinanalog" also refers to protected products of biological origin whichhave been modified after they have been produced, in particular, bychemical reaction or enzymic reaction.

The present invention relates to new vectors for the cloning andexpression of hirudin or a hirudin analog in a host cell, which vectorscontain the gene coding for hirudin or a hirudin analog and the elementsfor expression of this gene in the host cell.

The nature of the expression elements can vary depending on the natureof the host cell. Thus, in bacterial cells, the expression elements willcontain at least one bacterial promoter and a ribosome binding site(which constitutes that which will sometimes be referred to as thecoding region for the initiation of translation).

In general, the vectors according to the present invention will contain,in addition to the gene coding for hirudin, the following:

the origin of replication of a bacterial plasmid,

a promoter, especially all or part of a bacteriophage λ promoter, i.e.,P_(L), P_(R) or P'_(R) ;

a region coding for the initiation of translation, incorporating the ATGeither on the 5' end of the hirudin gene, or on the 5' end of thehirudin gene fused on the 5' side with another protein; this fusion isto make it possible to express a protein of high molecular weight whichis degraded to a smaller extent in E. coli.

The presence of an origin of replication for a plasmid is essential toenable the vector to replicate in corresponding bacterial cells. In thecase of E. coli, the origin of replication of plasmid pBR322 willpreferably be used. Plasmid pBR322 has, in effect, the advantage ofproviding a high copy number, and thus, increases the quantity ofplasmids producing the desired protein.

Among the bacteriophage λ promoters, the main leftward promoter,designated λ P_(L), will preferably be used. P_(L) is a powerfulpromoter responsible for the early transcription of λ.

It is also possible to use other bacteriophage λ promoters, inparticular, the rightward promoter P_(R) or the second rightwardpromoter P'_(R).

Although it is possible to use very varied translation initiationsequences, it is preferable to use all or part of the ribosome bindingsite of the bacteriophage λ protein cII, hereinafter referred to as λcIIrbs.

As will be shown below, it is also possible to use synthetic sequences,in particular, all or part of the sequence:

ATAACACAGGAACAGATCTATG.

The vector in question preferably contains, in addition, a transcriptionantitermination function encoded, eg., by the N gene of λ, referred toas λ N. In the presence of the gene N transcription product,transcription starting from P_(L) continues beyond most of the stopsignals.

This avoids the problems which are caused by a premature stopping oftranscription, which can occur when the cloned foreign genes possesssuch stop signals. In addition, it has been shown that expressionstarting from P_(L) is improved in an N⁺ environment.

In order to avoid the problems of toxicity and instability in thehost/vector system when continuous producing large amounts of a foreignprotein, it is necessary to provide for the control of the activity ofthe promoter by adjoining thereto all or part of an inducible expressionsystem, in particular, a temperature inducible system.

Control by temperature of the synthesis of the foreign protein ispreferably accomplished at the level of transcription, by means of atemperature sensitive repressor encoded in the host bacterium, forexample cI857, which represses P_(L) activity at 28° C. but isinactivated at 42° C. The repressor acts on the operator O_(L) which isadjacent to the promoter P_(L). Although in the above case a proportionof the temperature inducible expression system is an integral part ofthe host bacterium, it is possible to provide for this system to formpart of the vector itself.

The vector in question can also contain a gene for resistance to anantibiotic, for example ampicillin in the case of pBR322, but otherresistance genes can be used for resistance to tetracycline (Tet^(r)) orchloramphenicol (Cm^(r)).

The incorporation of such a marker is necessary for the selection of thebacteria containing the transformants carrying the plasmid according tothe invention during the cloning experiments.

The incorporation of a resistance gene permit the stability of theplasmid to be increased by imposing a selection pressure duringfermentation, and furthermore, facilitates the isolation of thetransformants.

For cloning, it is advantageous to have available a system whereby theinsertion of a foreign DNA into a plasmid can be detected.

By way of example, it is possible to provide in the cloning zone theN-terminal fragment of E. coli, β-galactosidase (lacZ'), by fusing thiswith the translation initiation region derived from λ cII. This and thisplaces the translation of the α fragment under the control of the cIIsequences.

The α fragment is complemented by the expression of the C-terminal ωfragment encoded in the host, and this leads to β-galactosidase activityin the cells. This β-galactosidase activity produces blue colonies inthe presence of a chromophoric substrate,5-bromo-4-chloro-3-indolyl-D-galactosidase (sic).

At 28° C., the P_(L) promoter is inactivated, the α fragment is notsynthesized and the colonies remain colorless. When the temperature israised to 42° C., the P_(L) promoter is activated, the α fragment issynthesized and the colonies turn blue.

The insertion of foreign DNA into the cloning sites located in thisdetection system prevents the synthesis of the β-galactosidase and henceleads to color-less colonies both at 28° C. and at 42° C. It is alsopossible to replace the lacZ' gene by other genes which permit detectionto be achieved.

Among the different hirudins which can be prepared and used, accordingto the invention, the following must be mentioned:

a) the hirudin corresponding to variant 2, known as HV1, the structureof which corresponds to that shown in FIG. 1 with or without the --SO₃ Hradical;

b) the hirudin corresponding to a modification of variant 1, in whichthe val--val N-terminal sequence has been replaced by ile-thr;

c) the hirudin corresponding to variant 1, known as HV2, the structureof which corresponds to that shown in FIG. 18b.

The structure of corresponding genes can be deduced from that of theamino acids, as will be shown in the examples. These genes can besynthesized by any one of the known methods for preparing synthetic DNA.

The hirudin in which the N-terminal structure corresponds to ile-thrwill preferably be used. Different structures of the corresponding genewill be seen in the examples.

The present invention relates to the cells, especially the bacteria and,in particular, the strains of E. coli, transformed by the vectorsaccording to the invention and using known techniques, some of whichwill be recalled in the examples.

Finally, the invention relates to a process for the preparation ofhirudin or its analogs, in which bacteria transformed as described aboveare cultured on a culture medium, and in which the hirudin or analogformed is then recovered.

The culture media employed are known to those versed in the art, andshould be suited to each strain cultivated. Culturing will preferably beperformed in the presence of the antibiotic against which thetransformed strain has become resistant.

The hirudin or its analogs can be purified or pre-purified from thefermentation mixture by being heated at an acid pH, in particular at50°-80° C. and at a pH between 1 and 3, especially at 70° C. and at pH2.8, with recovery of the supernatant in which the hirudin is present.

It is also possible to turn to account the affinity of hirudin forthrombin using a resin to which thrombin is bound, and passing themixture containing hirudin over such a resin. The hirudin is bound andthis can then be eluted with a solution containing a competitive agentfor hirudin.

The present invention finally relates to the hirudin or analogs thereofobtained by carrying out the process according to the invention, that isto say, the hirudin or analogs thereof of bacterial origin, but also thehirudins or analogs obtained from bacterial products by chemical orenzymatic reaction, for example, by chemical or enzymatic cleavage or,alternatively by chemical or, enzymatic reaction designed to bind an--SO₃ H radical.

In particular, the invention relates to the peptides containing all orpart of the following formula:

    __________________________________________________________________________    ATT                                                                              ACT                                                                              TAC                                                                              ACT                                                                              GAT                                                                              TGT                                                                              ACA                                                                              GAA                                                                              TCG                                                                              GGT                                                                              CAA                                                                              AAT                                                                              TTG                                                                              TGC                                    Ile                                                                              Thr                                                                              Tyr                                                                              Thr                                                                              Asp                                                                              Cys                                                                              Thr                                                                              Glu                                                                              Ser                                                                              Gly                                                                              Gln                                                                              Asn                                                                              Leu                                                                              Cys                                    CTC                                                                              TGC                                                                              GAG                                                                              GGA                                                                              AGC                                                                              AAT                                                                              GTT                                                                              TGC                                                                              GGT                                                                              AAA                                                                              GGC                                                                              AAT                                                                              AAG                                                                              TGC                                    Leu                                                                              Cys                                                                              Glu                                                                              Gly                                                                              Ser                                                                              Asn                                                                              Val                                                                              Cys                                                                              Gly                                                                              Lys                                                                              Gly                                                                              Asn                                                                              Lys                                                                              Cys                                    ATA                                                                              TTG                                                                              GGT                                                                              TCT                                                                              AAT                                                                              GGA                                                                              AAG                                                                              GGC                                                                              AAC                                                                              CAA                                                                              TGT                                                                              GTC                                                                              ACT                                                                              GGC                                    Ile                                                                              Leu                                                                              Gly                                                                              Ser                                                                              Asn                                                                              Gly                                                                              Lys                                                                              Gly                                                                              Asn                                                                              Gln                                                                              Cys                                                                              Val                                                                              Thr                                                                              Gly                                    GAA                                                                              GGT                                                                              ACA                                                                              CCG                                                                              AAC                                                                              CCT                                                                              GAA                                                                              AGC                                                                              CAT                                                                              AAT                                                                              AAC                                                                              GGC                                                                              GAT                                                                              TTC                                    Glu                                                                              Gly                                                                              Thr                                                                              Pro                                                                              Asn                                                                              Pro                                                                              Glu                                                                              Ser                                                                              His                                                                              Asn                                                                              Asn                                                                              Gly                                                                              Asp                                                                              Phe                                    GAA                                                                              GAA                                                                              ATT                                                                              CCA                                                                              GAA                                                                              GAA                                                                              TAT                                                                              TTA                                                                              CAA                                                   Glu                                                                              Glu                                                                              ILe                                                                              Pro                                                                              Glu                                                                              Glu                                                                              Tyr                                                                              Leu                                                                              Gln                                                   __________________________________________________________________________

This peptide is shown with the corresponding coding sequence which doesnot form part of the peptide.

The invention also relates to the variants of hirudin HV1 and HV2 asshown in FIG. 18.

The invention relates, finally, to pharmaceutical compositionscontaining hirudin or its analogs as an active principle.

These compositions can be administered intraperitoneally, intravenously,intramuscularly, subcutaneously, orally or by topical cutaneousadministration.

These compositions contain the excipients known in this field, andoptionally other active principles.

The present invention naturally includes other aspects, in particularcertain plasmids which will be described in the examples, as well as themutants and derivatives thereof and, in general, the processes offermentation of the transformed bacteria, as well as the productsthereby obtained.

Other characteristics and advantages of the invention will be more fullyunderstood on reading the examples below and the attached diagrams,wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of hirudin extracted from a wholeleech.

FIG. 2 shows the 48-mer oligonucleotide probe designed for thescreening.

FIG. 3 shows the hirudin sequence of the clone pTG717.

FIG. 4 shows an autoradiograph of the hybridization mixture between thelabeled probe and various pTG717 restrictions.

FIG. 5 shows the restriction map of the insert in pTG717.

FIG. 6 shows, in detail, the construction of the two vectors for theexpression of hirudin, pTG718 derived from pTG927 and pTG719 derivedfrom pTG951.

FIG. 7 shows in a graph, the changes in the hirudin activity withrespect to time in the E. coli extracts for the two vectors.

FIG. 8 shows the analysis of the labeled E. coli extracts containing thehirudin activity.

FIG. 9 shows the analysis of the same extracts as in FIG. 8, butfollowing heat treatment at an acid pH.

FIG. 10 shows the preparation of pTG907.

FIG. 11 shows the preparation of M13 TG 910.

FIG. 12 shows the preparation of pTG908.

FIG. 13 shows the preparation of pTG909.

FIG. 14 shows the preparation of pTG941.

FIG. 15 shows the preparation of pTG951

FIG. 16 shows a graph of hirudin activity induced in an E. coli TG900culture containing pTG720.

FIG. 17 shows a spectrum of analysis of the ³⁵ S-labeled proteins fromextracts of E. coli TG900 containing pTG720.

FIG. 18 shows amino acid sequences of HV1 and HV2.

FIG. 19 shows the single TaqI site in the cDNA of HV2 cloned in pTG717which is centered on amino acid 56.

FIG. 20 shows block 1 in the synthesis of the first 56 amino acids ofHV1.

FIG. 21 shows the synthesis of block 2 of the first 56 amino acids ofHV1.

FIG. 22 shows pTG726.

FIG. 24 is a graph showing the induction of antithrombin activity in anE. coli pTG726 culture grown at 30° C. to an optical density of 0.3 at600 nm, followed by induction at 37° C.

FIG. 25 is a graph showing the induction curve for pTG720 culture (FIG.25A) and pTG726 culture (FIG. 25B).

FIG. 26 shows the NdeI-PvuII fragment of pTG927 assembled with theHinfI-AhaIII fragment of pTG717 using adaptor oligonucleotides.

It is appropriate to note that the different nucleotide sequencesappearing in the diagrams must be considered to form an explicit part ofthe present description, these sequences not having been reproduced inthe body of the text so as not to encumber it needlessly.

Most of the techniques employed in the following examples are known tothose versed in the art, and some are described in the attachedreferences, only the special techniques will be discussed in the courseof the description. The characteristics of the strains used will only begiven by way of general information

Bacterial strains

The bacterial strains used in the context of the present invention areas follows:

TGE900, an E. coli strain having the following characteristics: su F hisilv bio (λcI857ΔBamΔHI).

N6437, an E. coli strain having the following characteristics: F⁻ hisilv gal⁺ Δ8proC⁺ : tn10 lac Δ m15 (λcI857ΔBamΔHI).

Jm103, an E. coli strain having the following characteristics: Δ(lac-pro) sup^(E) thi endA sbcB15 sttA rK⁻ nK⁺ /F¹ traD36 proAB⁺laci^(a) lacZΔm15.

The strains mentioned above were used because they were available, butit is obvious that it is possible to use other strains, provided thatthe strains have certain essential characteristics which are recalled inthe course of the detailed description.

The examples below essentially comprise the following stages:

1) the preparation of mRNA molecules and the formation of a cDNAlibrary;

2) the production of a probe;

3) the selection of the library by means of this probe and theidentification of a plasmid carrying the sequence coding for hirudin;

4) the production of a vector for the expression of the gene coding forhirudin;

5) a study of the results obtained.

Preparation of the RNA

Living leeches of the species Hirudo medicinalis are collected in theBordeaux region of France. They fasting for a minimum of 4 weeks andthen are decapitated, with the heads being immediately frozen in liquidnitrogen. The whole head RNA is extracted by grinding the heads into theform of a fine powder in liquid nitrogen. To 1 g of this powder, 5 ml ofan NETS buffer (10 mM Tris HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5%SDS) and 5 ml of redistilled phenol are added, both being preheated to95° C. The solution is then mixed by means of a vortex and centrifugedat 30,000 g, the aqueous phase is re-extracted with 1 ml of NETS bufferand the combined aqueous phases are re-extracted with 5 ml of freshphenol. The RNA is precipitated by adding two volumes of ethanol andcollected by centrifugation after the mixture has been left at -20° C.for over 4 hours. The dark brown pellet is dissolved in 2.5 ml ofdistilled water, 2.5 ml of 5M LiCl are added, and after being mixed, thesolution is left overnight at 4° C. The solution is then enriched with 5ml of 3M LiCl and centrifuged at 20,000 g for 10 minutes at 4° C. Thesupernatant is removed and the pale RNA pellet taken up in 1 ml ofwater. It is taken up in 0.25M NaCl and the RNA is precipitated with 2volumes of ethanol. The final RNA pellet is recovered by centrifugationand then dissolved and stored at -80° C. in distilled water. The RNAconcentration is measured by its absorption at 260 nm.

Polyadenylated messenger RNA is prepared using an oligo(dT)-celluloseand a standard procedure (22). The yield of mRNA representsapproximately 2 to 5% of the total starting RNA.

Preparation of complementary DNA and construction of the cDNA library inthe vector plasmid pBR 322

The process employed is exactly the same as that which led to theconstruction of the human liver mRNA library described in reference 23.The leech head mRNA molecules are copied into DNA form using anoligo(dT) "primer" and the enzyme reverse transcriptase. A second strandof DNA is synthesized using DNA polymerase, the hairpin is opened withS1 nuclease and the double-stranded cDNA is purified on a sucrosegradient. cDNA molecules of specified size are terminated at the 3' endwith small dC extensions using the enzyme terminal deoxytransferase, andare then united with a pBR322 vector having a polyG end cut by Pst1. Theplasmids obtained are used to transform E. coli strains (strain 1106),and the tetracycline-resistant transformants (obtained with anefficiency between 500 and 1,000 per ng of double-stranded cDNA) aregrown on L-agar plates containing 15 μg/ml of tetracycline. A cDNA clonelibrary representing 43,000 individual transformants is established, andthe cells are recovered from the colonies by scraping and resuspended inan L-broth containing tetracycline. A suspension is made in 50% strengthglycerol and stored at -20° C.

Production of the DNA probe for screening the library

In view of the fact that the amino acid sequence published for hirudin(FIG. 1) does not have methionine or tryptophan, that is to say, theamino acids which are encoded by a single codon, the protein sequencedoes not have regions which are particularly propitious for producingespecially short oligonucleotide probes which, as a general rule,constitute the strategy employed for identifying cloned DNA sequences(24). For this reason, it was decided to adopt the different techniqueof using a single but rather large oligonucleotide probe, and this madeit possible, in particular, to isolate the cDNA clone for human clottingfactor IX (23). Using this strategy, the amino acid sequence region isselected for in which the redundancy of the codons is limited to thechoice of the 3rd base (that is to say, arginine, leucine and serine areavoided), and the 3rd base is selected. The parameters in question forchoosing the 3rd base of this codon are known, and it is a question ofmaximizing the possibility of G/T interaction and avoiding hairpins andpalindromes in the probe sequence, and of course, of taking into accountthe known gene sequences for leeches.

Since no gene sequence has been published for leeches, it was necessaryto make use of the knowledge available in respect to the organismsclosest to the leeches from the evolutionary standpoint, and for whichmany published DNA sequences exist, i.e., insects. Accordingly thecoding sequences for insect DNA molecules were analyzed and a table wasdrawn up of the codons which arise most frequently. Together with otherparameters, this was used to design an oligonucleotide 48 bases long,corresponding to 16 amino acids, from position 34 to position 49 in thehirudin sequence. This sequence region shows redundancy only in thethird position of each codon.

This oligonucleotide, shown in FIG. 2, was synthesized chemically by thephosphodiester method, on an inorganic solid support (25), which hasalready been described for a reference 52-mer (23).

Screening of the cDNA library with the 48-mer oligonucleotide

The cDNA library formed from the leech head mRNA molecules is plated onL-agar plates containing 15 μg/ml of tetracycline with a colony densityof approximately 4,000 colonies per 13-cm plate, and the colonies aregrown to a size of 1 to 2 mm in diameter at 37° C. The colonies are thenwithdrawn onto nitrocellulose filters. The colonies on the maintainedplates are grown and the plates are then stored at 4° C. The filters areplaced on plates of L-agar containing 170 μg/ml of chloramohenicol andincubated overnight at 37° C. to amplify the plasmids in the bacterialcolonies. The filters are then treated using the standard procedure tolyse the bacterial colonies and bind the DNA to the nitrocellulose. Thefilters are then washed very thoroughly and then prehybridized in avolume of 100 ml of 6×SSC (1×SSC=0.15% NaCl, 0.015M trisodium citrate),5×Denhardts (1×Denhardts solution=0.02% Ficoll, 0.02% ofpolyvinylpyrrolidone, 0.02% of bovine serum albumin) 100 μg/ml of yeasttransfer RNA and 0.05% of sodium pyrophosphate.

An aliquot (30 pmol) of the 48-mer oligonucleotide is labeled with ³² P!at its 5' end by incubation with Γ ³² P!ATP in the presence of 15 unitsof polynucleotide kinase. The labeled probe is purified from free Γ ³²P!ATP by passage on DEAE-cellulose.

The labeled probe is hybridized on the filter at a dose of 20 ml of6×SSC, 1×Denhardts, 100 μg/ml of yeast tRNA, 0.05% of sodiumpyrophosphate at 42° C. with gentle agitation for 16 hours.

The filters are then washed in 6×SSC, 0.1% of sodium dodecylsulfate(SDS) at a temperature of 37° C., 42° C. and 50° C., and then subjectedto a final wash with 2×SSC, 0.1% SDS at 50° C. for 10 minutes. Thefilters are then dried and exposed on X-ray films. The colonies givingpositive results are identified from the labeled plates by comparing thepositive spots on the X-film with the master plates. One of theseclones, the plasmid of which will be referred to as pTG700, is confirmedas positive by a second hybridization with the labeled probe and iscultured in quantity, and the plasmid DNA is purified using standardprocedures.

Identification of pTG700 as containing the sequence coding for hirudin

The cDNA insert of pTG700 represents approximately 235 base pairs. ThisDNA fragment is isolated and transferred into the vector phage m13 mp8,and the DNA sequence of the insert is determined by chain termination(26). A portion of this sequence codes for a protein sequence which isvery similar to that of hirudin. This region of the sequence correspondsto the zone of the probe which is shown in FIG. 2.

In FIG. 2:

(a) corresponds to the hirudin sequence which appears in reference 2from glu 49 to gly 34,

(b) corresponds to the sequence of the 48-mer probe synthesized and usedfor the hybridization,

(c) corresponds to the sequence of the clone pTG700 in this region, thedots indicating the homologies, and

(d) corresponds to the amino acid sequence coded in this region ofpTG700.

It should be noted that the cDNA sequence in pTG700 is incomplete, as itencodes only 28 of the C-terminal amino acids of hirudin, in addition tothe 101 base of the 3' non-translated sequence preceded by a stop codonin phase. One of the differences relative to the published proteinsequence for hirudin is observed in this clone, since glutamic acidreplaces glutamine at position 49.

Isolation of clones of larger hirudin cDNA

Since the DNA sequence confirms that the pTG700 insert codes forhirudin, this insert is made radioactive with ³² P! using the known"nick translation" procedure and is used for a new test on the cDNAlibrary. Several other positive colonies are found and one of thesecontains a plasmid designated pTG717, which contains an insert ofapproximately 450 base pairs in length. Restriction analysis of thisinsert shows the presence of a Taq1 restriction site situated in theregion of the center.

For this reason, the purified fragment is digested with Taq1 and theresulting 5' and 3' fragments are linked in the sequencing vector m13cleaved with PstI/AccI. The construction of m13 contains each of thefragments which can be identified and subjected to sequential analysis.The result of this analysis is shown in FIG. 3. The DNA sequence of thepTG717 insert, minus the polyG/C ends introduced at the chain end duringthe cloning step of the procedure, is 379 bases long. Of the bases, 219code for a peptide whose sequence is very similar to that of thepublished hirudin sequence.

Various points must be noted in this sequence.

1) The amino acid sequence encoded by the fragment is not identical tothat of the published protein sequence for native hirudin. There is adifference of 9 amino acids between the hirudin sequence isolated frompTG717 and the published hirudin sequence. The following modificationsare noted: Val 1-ile; val 2-thr; gin 24-lys; asp 33-asn; glu 35-lys; lys36-gly; lys 47-asn; gln 49-glu and asp 53-asn. These modificationconstitute a major change in the sequence, since 7 of the modificationsinvolve a change in charge. The homology between the cloned codingsequence and the published protein sequence is thus 46/65, that is tosay, approximately 70%.

There are two possible reasons for the observed differences between thetwo amino acid sequences.

1 Species, subspecies and even individual animal variations can be seenbetween the exact sequence of the hirudin molecule. It is not possibleto verify whether the leeches used in this study are exactly of the samespecies or subspecies as those which were used to publish the amino acidsequence. Furthermore, it is possible that there exists in leeches notonly one but various forms of hirudin, with similar biological activitybut with variations in the fundamental amino acid sequence. In thiscontext, there should be noted to the differences in the results in theliterature relating to the N-terminal amino acids of hirudin(originally, ile had been found in the material from the heads, and thenval had been found in the animal as a whole, while more recently, ilewas indicated again as originating from the heads). Isoleucine ispresent in the pTG717 sequence at the position corresponding to theN-terminal end of the mature protein.

The concept of different forms of hirudin is supported by thepreliminary studies at the DNA level. When leech total DNA is extractedfrom ground frozen leeches using standard procedures, digested withvarious restriction enzymes, treated by electrophoresis on agarose geland then transferred to nitrocellulose and hybridized with the pTG717insert as a probe, a large quantity of fragments which hybridize isobserved.

Thus, the results in FIG. 4 are obtained in the following manner:

10 μg of the leech total DNA are digested with restriction enzymes andpressed to electrophoresis on a 1% agarose gel, transferred tonitrocellulose filters and hybridized with a ³² P!-labeled pTG717 PstIinsert. The filters are then washed thoroughly (0.1×SSC, 0.1% SDS, 65°C. and the hybridized bands are visualized using autoradiography.

Lane 1--DNA digested with EcoRI

Lane 2--DNA digested with HindIII

Lane 3--DNA digested with BamHI

Lane 4--DNA digested with BglII

The size of the EcoRI fragments in kb is indicated at the right.

With the Eco R1 digestion fragments, 6 fragments totaling 40 kb can beseen under very stringent washing conditions. Even if it istheoretically possible that this scheme represents a very broad mosaicgene, it is unlikely that a single small sequence coding for fewer than400 bases is distributed over 40 kb of genome. Furthermore, preliminaryexperiments with probes containing partial insert fragments suggest thatthis is not the case.

It was also observed that there was a difference in the sequences indifferent clones of hirudin isolated from the same cDNA library. Forexample, in pTG700, a lys is found in position 47 (FIG. 2), as in thepublished amino acid sequence, while in pTG717, there is an asn at thisposition.

For this reason, all these data are consistent with the concept ofhirudin genes of different structure, showing multiple forms at theprotein level.

A further point which is important is that the whole hirudin mRNAsequence is certainly not present in pTG717. In effect, open readingcontinues up to the 5' end of the clone, and there is no methionine inthis region. Since hirudin is a protein which is secreted from cells ofthe salivary glands, it is likely that it has, at its N-terminal end, aleader sequence (needed for secretion) and probably also a pro-sequence.The final active molecule would then be produced, as is the case formany zymogens and precursors, by proteolytic cleavage stages.

The size of the hirudin mRNA was determined, and confirms that the clonepTG717 is not a complete copy thereof. Hirudin total head RNA wassubjected to electrophoresis on agarose gel denatured with formaldehyde(27), transferred to nitrocellulose and hybridized with a PTG717 insert.A single, 640 base pair hybrid RNA species is observed. Since it isthought that pTG717 contains the whole 3' mRNA sequence (a polyA portionis observed), and likewise, two polyA addition sites which overlap,approximately 20 base from the polyA, a 160 bp 3' non-translated regionis seen (FIG. 3). This cDNA clone lacks 160 base pairs at the 5' end. Itcontains the remainder of the N-terminal end of the protein, themethionine of the initiator and a 5' non-translated region of the mRNA.It cannot be ruled out that hirudin is cut from the C-end in the form ofa larger precursor, which can code for other biologically activepeptides, although the maximum size of this hypothetical precursor wouldbe at least 110 to 120 amino acids (that is to say, approximately twicethe size of mature hirudin).

It was not possible to generate cDNA clones longer than pTG717, and itis thought that a secondary structure in the 5' end of the mRNA preventsreverse transcription of the mRNA beyond this point. Analysis of thegenomic sequence of hirudin should provide data on the 5' end of thisgene.

The amino acid sequence of hirudin encoded by the clone pTG717 wasadapted so as to be expressed in E. coli cells, in the manner to bedescribed below.

Expression of hirudin in E. Coli

From the restriction analysis of pTG717 (FIG. 5), it is clear that thewhole of the amino acid coding sequence is present in the form of a 225bp HinfI-AhaIII fragment. This fragment is isolated by restriction andinserted in the plasmid expression vectors pTG951 and pTG927, usingsynthetic oligonucleotide adaptor molecules. The adaptors constitute the7 amino acids which are removed from the N-terminal end by HinfIrestriction, and the initiator methionine fragment and the site for therestriction endonucleases NdeI or BglII, which are required forinsertion in the expression vectors, are added.

The two amino acids after the met initiator are replaced, that is tosay, the ile-thr fragment which appears in the pTG717 cDNA clone isreplaced by val--val of the published hirudin protein sequence.

Although the pTG717 cDNA clone contains the ile-thr sequence instead ofthe val--val sequence of the published sequence for hirudin, it wasinitially decided to express the molecule with a val--val sequence atthe N-terminal end. There are two reasons for this choice:

the cDNA clone is certainly incomplete, there probably being a secondarystructure in the mRNA which prevents transcription; this makes itpossible for the 5' end of the cDNA sequence to be incorrect, takinginto account the transcription and cloning of "arti-facts"; and

it is generally accepted that the N-terminal end of the active hirudinmolecule extracted from the whole animal begins with val--val (8, 11).

The expression vector pTG951 is a plasmid expression vector designed toexpress the interferon-Γ gene, the synthesis of which is recalled at theend of the present description.

In practice, it contains, in essence, the bacteriophage leftwardpromoter P_(L) controlled by a repressor encoded by the temperaturesensitive host C1857, followed by the N gene of λ, intact or truncated.A site for binding synthetic ribosomes is then present, this beingdesigned to give optimal binding of ribosomes. A single BglIIrestriction site is present located between the ribosome binding siteand the ATG of the sequence coding for interferon-Γ. This vector isdigested at the BglII site and at the single PvuI site downstream of thesequence coding for interferon-Γ, and is then recovered byelectrophoresis on agarose gel. This vector fragment is combined with anadaptor oligonucleotide assembly and the hirudin sequence containing theHinfI-AhaIII fragment, as described in FIG. 6.

The expression vector pTG927 is closely related to the expression vectorpTG908 described at the end of the present description. It differs onlyin that it contains an additional SalI-PvuII fragment originating fromthe tetracycline gene of pBR322. The expression vector pTG927 containsthe promoter P_(L), an intact N gene and the ribosome binding site ofthe Γ CII protein, then the ATG and a sequence coding for the lacZfragment of β-galactosidase. An NdeI site is found on the ATG of thiscoding sequence. This vector is digested with NdeI and PvuII and thevector fragment is purified. It is used together with anotheroligonucleotide adaptor combination and the HinfI-AhaIII fragment ofhirudin to construct the second hirudin expression vector shown in FIG.6. The two expression vectors are designed to express the native hirudinmolecule (with a val--val sequence at the N-terminal end immediatelyafter the ATG initiator).

In the adaptor oligonucleotide, the codon of the 3rd base is chosen topromote a high degree of translation and to avoid the formation ofsecondary structures in the mRNA at the level of this region.

The two vectors are assembled in the following manner: theoligonucleotides are phosphorylated at the 5' end with polynucleotidekinase, and then treated in an equimolar mixture (after 10 minutes'heating at 65° C., for 15 hours at 15° C.). The vector and the hinrudinfragment are provided in amounts designed to give mol ratios ofvector/hirudin fragments/adaptor fragments of 1:20:50, and the mixtureis ligated with T4 DNA ligase. The ligation mixture is used to transformE. coli strain TG900 and the transformants carrying the plasmids areselected on an agar plate containing ampicillin (100 ug/ml), and thosewhich contain the sequence coding for hirudin are identified byhybridization of the colonies using a labeled pTG717 insert as probe.From a large number of positive clones, 6 of each, as well as 1 negative(parent strain), are selected and grown in an L-broth liquid medium at30° C. to an optical density of 0.3 at 650 nm. Expression starting fromthe P_(L) promoter is then induced by increasing the temperature to 37°C. and an incubation which is maintained for 6 hours. The cells are thenharvested by centrifugation suspended in 1/5 of the volume of TGE (25 mMTris HCl, pH 8, 50 mM glucose, 10 mM EDTA) and then ground bysonication. After clarification of the extracts by centrifugation, analiquot of the supernatant is tested for its antithrombin activity.Significant levels of antithrombin activity appear in 5 of the 6constructions with pTG927 and in 4 of the 6 constructions with pTG951,which also show activity although this is weaker.

The controls do not show any significant activity. Analysis of the DNAsequence of the clones giving positive results by direct sequencing ofthe plasmids shows that the expected sequence is present in all theconstructions which show activity.

Two clones showing positive expression, the clone pTG719, derived frompTG951, and the clone pTG718, derived from pTG917, are analyzed for 6hours of induction. After growth of the culture at 30° to an opticaldensity of 0.3, expression is induced by raising the temperature to 37°C., and an aliquot is withdrawn every hour for 6 hours from the extractsas prepared above, and the hirudin activity is measured (29). It is seenfrom the results (FIG. 7) that the clone pTG719 shows an initial growthfollowed by a plateau of activity after 3 to 4 hours, which is at thelevel of 370 U/l at an optical density of 650.

In contrast, the clone pTG718 shows greater activity, which continues toincrease throughout this entire induction period.

The level of activity obtained after 6 hours is approximately 5 timesgreater than that of the clone pTG719, and represents a total activityof 7300 μ/l of culture. If this hirudin recombinant shows specificactivity similar to that of the natural val--val product, the productobtained in the most active extracts would correspond to approximately 1mg/l of culture.

Property of the hirudin obtained

In order to characterize the hirudin obtained from E. coli, analysis wasperformed directly on culture samples after induction, which were heatedto 70° C. for 15 min or heated to 70° C. after reduction of the pH to2.8 with HCl. In the latter case, the pH of the extract was brought backto neutral before being assayed. Native hirudin treated in the samemanner shows no loss in activity, as could be expected in light ofpublished material on the properties of the molecule (20). In the caseof the extract, no loss in activity is seen after this treatment, infact, after the treatment, an approximately 2-fold increase in activityis observed. This can reflect either the degradation or the inactivationof constituents of the extract which inhibited the action of hirudin, orpossibly the rather low pH and the heating stage may, in some cases,provide for a more complete re-forming of the disulfide bridges whichare needed for hirudin to show maximum activity. The control extractsshow no activity, either before or after the treatment.

When the bacterial extracts are preincubated with thrombin bound to aSepharose resin, and the thrombin-Sepharose is removed bycentrifugation, virtually all the initial hirudin activity is removedfrom the extract. The hirudin obtained according to the presentinvention appears to bind very effectively to the thrombin-Sepharoseresin, and this makes it possible to envisage a possible means ofpurifying this bacterial hirudin.

Bacterial cultures of cells containing the expression vector pTG718 arecultured to an optical density of 0.3 at 30° C. and then induced at 37°C., and aliquots are withdrawn every hour and labeled, on minimalmedium, with 100 μCi/ml of ³⁵ S!methionine. The bacteria are collectedby centrifugation and the combination of labeled bacterial proteins isanalyze by electrophoresis on SDS-polyacrylamide gel, followed byfluorography.

At least two polypeptide induced in significant quantity by the vectorconstruction can be observed at approximately 6 to 8,000 daltons (FIG.8--lanes 5 to 10).

When the labeled materials from non-induced pTG718 cultures and fromcultures at 3 and 5 hours after induction are treated at 70° C. and atpH 2.8 for 15 min, centrifuged to remove the denatured proteins andanalyzed on an SDS-polyacrylamide gel, it is clear that the bulk of thelabeled E. coli proteins are removed from the sample (FIG. 9--lanes 1and 2). This procedure gives a very satisfactory purification of the twobands of low molecular weight which are induced in the hirudinexpression vectors.

All the hirudin activity is found in the supernatant.

Preparation of the vector plasmids pTG951 and pTG927

These vector plasmids have been used for cloning interferon-Γ or havebeen derived from plasmids which participate in the preparation of suchvectors. This synthesis will be recalled briefly below in relation tothe attached figures, wherein:

FIG. 10 shows the preparation of pTG907

FIG. 11 shows the preparation of M13 TG 910

FIG. 12 shows the preparation of pTG908

FIG. 13 shows the preparation of pTG909

FIG. 14 shows the preparation of pTG941

FIG. 15 shows the preparation of pTG951.

The preparation of these vector plasmids comprises essentially:

a) the preparation of pTG908, which vector contains P_(L), N and CIIrbs; and

b) the preparation of pTG951, which vector contains a binding site forsynthetic ribosomes.

Preparation of pTG907 (FIG. 10)

The parent plasmid used is plasmid pBR322. However, the latter has thedisadvantage of having a PstI restriction site within the amp^(R) gene,since a site of the same nature will be used subsequently in the cloningzone as a single restriction site. Therefore, it is hence appropriate tomake cause this PstI restriction site to disappear, using a mutant ofplasmid pBR322, plasmid pUC8, in which the gene for resistance toampicillin does not have a PstI restriction site (this site has beenremoved by mutation in vitro). pBR322 is marketed in particular byBethesda Research Laboratories and pUC8 is described in the paperdesignated as reference 30.

For this purpose, the 1,669 bp PvuI-PvuII fragment of pBR322 isexchanged with the analogous PvuI-PvuII fragment of plasmid pUC8. Tocarry out this exchange, plasmids pBR322 and pUC8 are treatedsuccessively with PvuI and PvuII, and are then circularized by theaction of a ligase.

Plasmid pTG902, which no longer has a PstI restriction site and whichhas also lost the NdeI restriction site present originally in pBR322(not shown in the Figure), is thereby obtained. Furthermore, plasmidpTG902 carries a 50 kb fragment corresponding to the laci' sequence inwhich the PvuII site is present.

The P_(L) promoter and the λ N gene (which originates from phage λ, thegene λ N coding for a transcription antitermination function) areisolated from plasmid pKC30 and inserted in pTG902, as shown in theattached FIG. 10, by treatment with EcoRI, S1, BamHI for pTG902 and bytreatment with PvuI, S1, BamHI for pKC30 with ligation.

One of the plasmids obtained after transformation of strain TGE900,pTG906, is treated so as to remove the PvuII-SalI segment. For thispurpose, pTG906 is treated successively with SalI, S1 nuclease, PvuIIand the ligase. pTG907 is thereby obtained.

Preparation of M13 TG910 (FIG. 11)

The λ cIIrbs "ribosome binding region" (which likewise originates fromphage λ) is then inserted in the form of an AvaI-TaqI fragment into thebeginning of the lacZ' gene (α fragment of β-galactosidase), which hasbeen cloned in the phage M13 known as M13tg110. This strategy enables asimple functional test to be carried out for rbs, i.e., the productionof the lacZ' protein, and consequently allow blue plaques to be obtainedin the presence of IPTG and Xgal. This also permits rapid sequencing ofthe construction using the so-called dideoxy method.

After selection in competent bacteria, a resultant clone M13tg910 isthereby obtained, the overall structure of which is shown at the bottomof the Figure.

Preparation of pTG908 (FIG. 12)

The cIIrbs-lacZ' fragment of phage M13tg910 is transferred to the vectorplasmid pTG907 prepared previously.

For this purpose, the EcoRI, BamHI and AvaI sites upstream from cIIrbsare removed and a BglII site is then inserted.

Under these conditions, cIIrbs can be withdrawn in the form of aBglII--BglII fragment and placed in the BamHI site downstream from theP_(L) promoter and from the λ N gene of pTG907.

Phage M13tg910 is digested with EcoRI and then treated with Ba131, thenfollowed by Klenow polymerase. The fragments obtained are then subjectedto the action of the ligase in the presence of non-phosphorylated BqlIIadaptor. The ligation mixture obtained is used for transformingcompetent JM103 cells.

The blue areas are then selected. These clones are then analyzed inorder to verify that they contain the BglII site and that they no longerhave an EcoRI or BamHI site upstream. Clones such as M13tg912, thestructure of which is shown, are thereby obtained.

The treatment with Ba131 produced a 101 bp deletion, eliminating theEcoRI, BamHI and AvaI sites; as well as the lac ATG and lacShine/Dalgarno sequences. The BglII site introduced is situatedapproximately 100 bp upstream of the cII ATG and 10 bp downstream ofP_(lac).

The BamHI-SphI fragment of pTG907, the BglII/HpaI fragment carryingcIIrbs and lacZ', and the phosphorylated adaptor are prehybridized in amol ratio of 1:2:1, and then treated with T₄ ligase. Aliquots are usedfor transforming competent cells of strain 6150 at 3° C.

The cells of interest are identified by selecting the transformants witha ³² P-labeled cIIrbs/lacZ' fragment, and the construction obtained isconfirmed by an enzyme restriction study.

In order to have an initial indication that the different elements ofthe expression system are behaving as desired, the plasmid obtained,pTG908, is transferred into an N6437 host strain which possesses bothc1857 and the ω fragment of β-galactosidase, which complements the αfragment which is encoded by the plasmid.

The transformants obtained, placed on a dish containing IPTG+Xgal, arecolorless at 28° C. and then turn blue after about 30 minutes when theyare transferred to 42° C.

Before being used for cloning hirudin, this vector was adapted to clonehuman interferon-Γ, IFN-Γ, and in fact, for cloning hirudin the natureof the cloned intermediate protein is of no importance, but the vectorswere produced according to the scheme below.

Analysis of the IFN-Γ nucleotide sequence for the restriction sitesreveals an EcoRII site 8 bp downstream of the starting point of themature protein and an Sau3A site 285 bp downstream of the stop codon,and this enables virtually the entire sequence coding for the matureprotein to be isolated on an EcoRII-Sau3A fragment. The IFN-Γ cloneobtained from a library is referred to as pTG11.

Construction of pTG909 (FIG. 13)

A synthetic adaptor molecule is first used, which allows:

a) joining to be accomplished between the EcoRII and NdeI ends,

b) the 8 bp, missing with respect to the sequence which codes for matureIFN-Γ, to be introduced, and

c) the cIIrbs ATG starting codon to be reconstituted, so that thesequence which codes for the mature IFN-Γ protein is translated withoutfused amino acids, with the exception of the F-met initiator.

This adaptor is chemically synthesized and its structure is shown in theFigure.

pTG11 is digested with EcoRII and Sau3A, and pTG908 with NdeI and BamHI.

The appropriate fragments are purified on gel, mixed with an equimolaramount of the adaptor, prehybridized and ligated. The mixture is usedfor transforming competent TGE900 cells, and the transformants areselected by hybridizing a nick-translated, ³² P-labeled pTG11 PstIinsert with the transformants.

13 clones are selected and monitored by mapping, and one of these,pTG909, is verified by sequencing.

Construction of the vector pTG941 (FIG. 14)

pTG909 contains 2 NdeI sites, one at the starting codon of IFN-Γ and theother 22 bp downstream from the IFN-Γ sequence.

The region between these sites, which is the region which codes for thefirst 7 amino acids of IFN-Γ, is removed by treatment with NdeI, andreplaced by a synthetic oligonucleotide which is shown in the Figure.

This reaction destroys the NdeI site downstream and reconstitutes theNdeI site upstream, while introducing a BamHI site which is unique. Thevector pTG941 is thereby obtained.

Construction of pTG951 (FIG. 15)

FIG. 15 shows schematically the construction of pTG951, which is derivedfrom pTG941, in which the fragment containing the cIIrbs has beenreplaced by a synthetic sequence based on the sequence of thetranslation initiation region of the E. coli lac operon, designated E.coli lac operon rbs. This synthetic oligonucleotide is inserted at theHgaI site between the single NdeI site of the starting codon of thesequence which codes for IFN-Γ the ClaI site which is inserted in the Ngene.

As a result, upon treatment with NdeI and ClaI, plasmid pTG951 now onlycontains a truncated N gene (a stop codon in phase with the translationof the N gene is situated immediately upstream from the new rbs site)and is devoid of the transcription terminators tL1 and tR1 present inpTG909 and pTG941.

The principal results are recorded in the Table below

    __________________________________________________________________________    NAME                                                                              PROMOTER                                                                             RBS RBS SEQUENCE AND JUNCTION WITH THE SEQUENCE                    __________________________________________________________________________                   fmet           cys                                                                              tyr                                                                              cys                                                                              gln                                                                              asp                                                                              pro                              PTG909                                                                            PL     cII TAAGGAAGTACTTACATATG                                                                         TGT                                                                              TAC                                                                              TGC                                                                              CAG                                                                              GAC                                                                              CCA                                             fmet           cys                                                                              tyr                                                                              cys                                                                              gln                                                                              asp                                                                              pro                              pTG941                                                                            PL     cII TAAGGAAGTACTTACATATG                                                                         TGC                                                                              TAC                                                                              TGT                                                                              CAG                                                                              GAT                                                                              CCC                                             fmet           cys                                                                              tyr                                                                              cys                                                                              gln                                                                              asp                                                                              pro                              pTG951                                                                            PL     SYNTH                                                                             CACAGGAACAGAGATCTATG                                                                         TGC                                                                              TAC                                                                              TGT                                                                              CPG                                                                              GAT                                                                              CCC                                         (lac)                                                                             BglII                                                          __________________________________________________________________________

The examples below are intended to illustrate the preparation of thevariant HV2, modified at its N-terminal

In the attached figures:

FIG. 16 shows a curve of the hirudin activity induced in an E. coliTG900 culture containing pTG720; and

FIG. 17 shows a spectrum of analysis of the ³⁵ S-labeled proteins fromextracts of E. coli TG900 containing pTG720.

Preparation of modified HV2

The construction of plasmid pTG720 expressing hirudin according to theinvention is obtained according to the same process as plasmid pTG718described above, starting with pTG717 and pTG927.

The NdeI-PvuII fragment of pTG927 is assembled with the HinfI-AhaIIIfragment of pTG717 by way of adaptor oligonucleotides, described below,in order to reconstitute the ile-thr sequence at the N-terminal end ofthe hirudin. ##STR1##

The ligation mixture is used for transforming E. coli TG900, and thetransformants containing plasmids are selected on an agar-L platecontaining 100 ug/ml of ampicillin. The constructions which contain thehirudin sequence are identified by hybridizing the colonies, using alabeled pTG717 insert as probe.

The DNA sequence of the final plasmid is monitored by direct sequencingof the DNA in the expression plasmid.

Expression of hirudin activity by pTG720

E. coli TG900 cells containing plasmid pTG720 are grown on LB mediumplus 50 μg/ml of ampicillin at 30° C. to an optical density at 600 of0.3.

The cell cultures are then transferred at 37° C. to induce transcriptionfrom the P_(L) promoter.

1 ml aliquots are withdrawn hourly and the density at 600 nm ismeasured; then the cells are collected by centrifugation.

The centrifugation pellet is resuspended in 200 μl of TGE (25 mM TrisHCl, pH 8.0, 50 mM glucose, 10 mM EDA) and the cells are lysed bysonication.

After clarification, the supernatant is collected and the antithrombinactivity is measured, either by the coagulation test or by colorimetricassay of the inhibition of cleavage of the substrate,tosylglycylprolyl-arginine 4-nitroanilide acetate (Chromozym TH,Boehringer Mannheim GmbH), by a standard solution of thrombin.

The reaction is performed in a reaction volume of 1 ml, using 13 μMsubstrate in a buffer composed of 100 mM Tris HCl, pH 8.0, 0.15M KCl and0.1% of polyethylenes glycol 6000.

The reaction with 0.25 U of thrombin is followed or 2 minutes using aspectrophotometer at 405 nm, and the rate of reaction is measured fromthe slope of the increase in optical density.

Standard hirudin or unknown extracts are added to this thrombin reactionmixture to determine the extent of inhibition or antithrombin activity.

FIG. 16, attached hereto, shows the effect of induction of antithrombinactivity in a culture of cells containing pTG720, expressed asantithrombin units for an optical density of 600 per liter of culture,the induction taking place over a period of 6 hours.

The broken line shows the growth curve of the E. coli cells measured atan optical density of 600 during the same period.

As a result of the induction, activity of the hirudin type, showing asignificant level, is observed.

Control lysates of the cultures containing the plasmid without thehirudin sequence show no activity.

When these bacterial lysates are heated to 70° C. for 15 minutes afteracidification to pH 2.8 with HCl, a considerable amount of protein isdenatured and precipitated. When the latter is removed by centrifugationfrom the cooled extract, and the supernatant is neutralized by adding aTris HCl buffer (final concentration 100 mM, pH 8.0), at least 100%, andfrequently more, of the starting activity reappears in the supernatant.

In a typical experiment, 130% of the starting activity reappears in thesupernatant.

No residual activity is found in the precipitated material in thepellet.

When a bacterial extract, heated and acidified after cooling,centrifugation and neutralization (200 μl containing 5 ATU of hirudin),is incubated for 15 minutes at 37° C. with 100 μl of a 50% strengthslurry of thrombin covalently coupled to a Sepharose resin (prepared bystandard procedures) and when the Sepharose-thrombin is removed bycentrifugation, no activity of the hirudin type can be found in thesupernatant.

Thus, the hirudin produced by pTG720 has the same general properties asthat of the native molecule and as that which is obtained by pTG717 andpTG718.

The polypeptides specifically induced in the pTG720 culture, afterlabeling with ³⁵ S!methionine, resolution by electrophoresis onpolyacrylamide gel and visualization by fluorography, are shown in FIG.2. A series of low molecular weight (5 to 10,000 daltons) polypeptidesare more especially induced.

In FIG. 17,

lane 1 shows the non-induced cells,

lane 2 the induction at 0 hour,

lane 3, 1 hour's induction,

lane 4, 2 hours' induction,

lane 5: molecular weight markers,

lane 6, 3 hours' induction,

lane 7, 4 hours' induction,

lane 8, 5 hours' induction,

lane 9, 6 hours' induction,

lane 10, 7 hours' induction.

The examples which follow are intended to illustrate the preparation ofhirudin HV1.

Study of the HV1 gene and strategy of synthesis

The strategy of synthesis for preparing the gene for hirudin HV1comprises various stages.

First, since there is no difference in amino acids between HV1 and HV2after amino acid 53, as is seen in FIG. 18, and since there is a singleTaqI site in the cDNA of HV2 cloned in pTG717 which is centered on aminoacid 56 (FIG. 19), the DNA sequence of the variant HV1 after amino acid56 can be provided by the TaqI-PstI fragment of pTG717.

For this reason, only the DNA coding for the first 56 amino acids of HV1have to be chemically synthesized.

This DNA was synthesized in two separate blocks. The first, shown inFIG. 20, begins with an EcoRI cohesive site which is primarily intendedfor cloning, immediately followed by an NdeI site which incorporates theATG initiation codon before the sequence which codes for HV1. Thecomplete gene can be withdrawn, using the NdeI site at the 5' end, forinsertion in an expression vector in E. coli. This portion is followedby a DNA extension coding for amino acids 1 to 32 of HV1, and can beterminated by a BamHI cohesive end, since amino acids 31 and 32 are glyand ser, and can be encoded by a BamHI site, as shown: ##STR2##

This synthetic DNA portion which possesses 109 bp, is assembled bycondensing its constituent oligonucleotides, and is then placed byligation in phage M13mp8 cut with EcoRI/BamHI in order to lead to theconstruction M13TG724. Since the synthetic DNA is present in thepoly-linker region of phage M13, it can be immediately sequenced toverify that this first synthetic block is correctly assembled.

The second synthetic block corresponds to amino acids 33 to 56, and islimited at one end by a BamHI cohesive site and at the other end by aTaqI site (FIG. 21). This 69 bp synthetic block is again assembled fromits constituent oligonucleotides, and then incorporated with theTaqI-PstI fragment originating from pTG717 into M13TG724 cut withBamHI/PstI, as shown in FIG. 21. This leads to a phage M13TG725 whichcontains the sequence coding for the complete HV1. As above, the correctassembly of this construction can be immediately verified by sequencing.

The following stage comprises the transfer of the NdeI-AhaIII fragment,which begins with the ATG of the hirudin sequence and terminates in anon-translated region at the 3' end, into plasmid pTG927 cut withNdeI/PvuII. This expression vector is identical to that which was usedfor constructing the hirudin HV2 expression vector pTG720, and itsstructure and construction have already been described. The finalexpression vector coding for the variant HV1 is known as pTG726, and isshown in FIG. 22.

The exact sequence of the oligonucleotides used for constructing thesetwo blocks is shown in FIG. 23. Block 1 extends from the EcoRI site tothe BamHI site, and is composed of 8 oligonucleotides having sizes whichrange from 22 to 32 bases. Block 2 extends between the BamHI site andthe TaqI site, and is composed of 6 oligonucleotides having sizesranging from 19 and 30 bases.

The oligonucleotides are synthesized by a manual phosphotriesterprocedure on a silica support (reference 31) and are purified using HPLCtechniques or elution from polyacrylamide gel.

The exact sequence of the oligonucleotides used for the syntheticportion of the gene is chosen having with the following parameters inmind:

a) the choice of codons, where possible, is that for the genes which areexpressed at very high levels in E. coli; this choice is made usingpublished data (ref. 32, 33);

b) computer analysis of each of the oligonucleotides, individually andthen in complete sequence, so as to eliminate structures which can form"hairpins";

c) the choice of N-terminal the end of the hirudin molecule correspondsto the preferred use of certain codons to obtain the bases in certainpositions in this region, where it has been shown that these bases wereimportant for a high level of expression of foreign proteins in E. coli.

Assembly of the synthetic gene First synthetic block

The oligonucleotides forming this block, 1-8 (FIG. 23), arephosphorylated at their 5' end with polynucleotide kinase under standardconditions, except for the two end oligonucleotides 1 and 8. This isintended to avoid the formation of dimer or polymer of the syntheticblock in the following ligation stages. 500 picomoles of each of theoligonucleotides are subjected to the action of kinase, using 2 units ofpolynucleotide kinase in a final 25 μl volume of 60 mM Tris HCl, pH 7.5,10 mM MgCl₂, 8 mM dithiothreitol, also containing 3.3 pmol of Γ-³²P!ATP, the specific activity of which is 5000 Ci/mmol. After incubationfor 15 minutes at 37° C., the oligonucleotides are then completelyphosphorylated by adding 5 mmol of cold ATP.

After incubation for a further 15 minutes at 37° C., theoligonucleotides are purified by electrophoresis on 20% polyacrylamidegel, performed under denaturing conditions. The labeled oligonucleotidesare detected by autoradiography, the appropriate regions of the gel areexcised and the oligonucleotides are eluted with water during incubationovernight at 37° C.

The oligonucleotides are then charged onto columns of DEAE-cellulose,eluted with a 1M triethylammonium bicarbonate buffer, pH 8, andlyophilized.

For oligonucleotides 1 and 8, which were not subjected to kinase and notlabeled, gel purification is carried out as above but theoligonucleotides are detected by UV absorption.

The complementary fragments (1+5, 2+6, and the like) are mixed, usingequivalent amounts 100 picomoles of each of the oligonucleotides in afinal 50 μl volume of 66 mM Tris HCl, pH 7.5, 6 mM MgCl₂, 100 mM NaCl,0.5 mM spermidine and 8 mM dithiothreitol. These mixtures are heated to100° C. and cooled slowly to 37° C. for 2 hours. The solutions are mixedto give hybrids with 4 oligonucleotides in 100 μl. The mixtures arefinally united and the 8 oligonucleotides are left to form pairsovernight at 37° C. in a final volume of 200 μl. 0.005 picomoles of thepaired oligonucleotides are ligated with 25 ng of M13mp9, digested withBamHI/EcoRI and purified on gel, in a final 20 μl volume of a ligationmixture containing 66 mM Tris pH 7.5, 6.6 mM MgCl₂, 10 mMdithiothreitol, and 0.5 mM ATP.

The ligation is continued at 15° C. for 24 hours, followed by ligationfor 24 hours at 4° C. The ligation mixture is then used for transformingE. coli JM103. From many colorless plaques obtained by transformation, 8candidates are selected, single-stranded DNA is prepared from the phagesand this is subjected to direct DNA sequencing by the dideoxy chaintermination method (reference 34). From these candidates, two are foundto contain the correct assembly of oligonucleotides corresponding toblock 1 of FIG. 23, and one of these is designated as M13TG724 and usedin the following stage.

Second synthetic block and assembly of the whole gene

To assemble the second synthetic block, the strategy used wasessentially the same as that described above, with the exception thatthe oligonucleotide constituents are not pre-purified before the pairingstage, but instead are subjected to kinase (except for the terminaloligonucleotides 9 and 14; see FIG. 23) and then directly paired. Thepairing conditions are the same as those described above, with 100picomoles of each of the oligonucleotides in a final volume of 150 ul.

After the pairing stage, the mixture is charged on a 2% agarose gel (lowmelting point agarose) and then subjected to electrophoresis. Theoligonucleotides are detected by staining with ethidium bromide and thebands corresponding to the assembled block (69 bp) are cut from the geland eluted by standard procedures.

The second synthetic block (2 ng) is then mixed with 50 ng of phageM13TG724, cut by PstI/BamHI and purified on gel, and 2 ng of a TaqI-PstIfragment of pTG717 purified on gel. The combination of these elements isassembled in a 20 μl volume of 66 mM Tris HCl, pH 7.5, 6.6 mM Mg Cl₂,and heated to 65° C. for 5 minutes, and there are added DTT to aconcentration of 10 mM, ATP to a concentration of 0.5 mM and 5 units ofT₄ ligase. The ligation is continued for 16 hours at 15° C. and theligation mixture is then used for transforming E. coli JM103.

From the colorless plaques among the transformants, 12 are chosen, whichare selected for preparing a single-stranded phage for directsequencing. Furthermore, the phage is also prepared in double-strandedform (reference 35) to study the existence of a single BamHI site whichhas to be present in the correctly assembled recombinates. The majorityof these clones contain both the BamHI site and the DNA sequencecorresponding to the correct assembly of the whole gene coding for HV1.One of these, designated M13TG725, is chosen.

Transfer of the HV1 gene to the expression vector

The final stage for creating the vector plasmid capable of expressingthe HV1 protein consists of transferring the 248 bp NdeI-AhaIII segmentof M13TG725 into pTG927 cut by NdeI/PvuII (FIG. 22). However, since thereplicative form of the phage with this type of digestion leads to asecond M13 fragment, which is practically the same size but which clonesmore efficiently in the expression vector than the desired fragment, itis necessary first to prepare an AvaII-BglII fragment (1.71 kb) whichcontains the whole of the hirudin HV1 sequence, and then to digest itwith NdeI/AhaIII. This digestion product, without further purification,is ligated in the expression vector pTG927 after cutting withNdeI/PvuII. Among the transformants of E. coli TGE900, the correctconstruction pTG726 is identified by the presence of a single BamHI sitederived from the sequence of the HV1 gene, and then by direct DNAsequence analysis.

Expression of the biological activity of hirudin HV1 with pTG726

The expression vector pTG726 contains a temperature inducible promoter,the P_(L) promoter, the major leftward promoter of bacteriophage λ.Since this promoter is blocked by a temperature sensitive repressorencoded by the host, the hirudin HV1 gene is not transcribed duringgrowth at 30° C. However, when the temperature is raised above 37° C.,the transcription of this gene is induced.

FIG. 24 shows the growth curve and the curve for induction ofantithrombin activity in an E. coli pTG726 culture grown at 30° C. to anoptical density of 0.3 at 600 nm, followed by induction at 37° C.

The hirudin activity is measured by the capacity of the sonicatedextracts of the bacterial cells to inhibit bovine thrombin activity withrespect to its capacity to cleave chromogenic substrates.

It is clear that significant amounts of hirudin are induced in thepTG726 cultures. Approximately 3 to 4,000 antithrombin units/OD/liter ofculture, but this activity declines rapidly as time proceeds. Thiseffect is readily reproducible, with a peak of activity 3 hours afterinduction, followed by a stage of decline.

The nature of this induction curve is very characteristic andreproducible, and differs significantly from those which have beenobserved previously with the expression vector pTG720 which, oninduction, expresses the hirudin variant HV2. This variant is inducedmuch more slowly (FIG. 25A) with a latency period of approximately 2hours, the activity increasing to reach virtually the same level as thatof pTG726 (FIG. 25B) but then remaining constant without any indicationof a decline. Since the two hirudin variants were produced using exactlythe same expression vector in exactly the same E. coli TGE900 host cell,this difference in induction and stability must definitely be connectedwith differences in the primary structures between HV1 and HV2. Thismay, furthermore, reflect a difference in the resistances to proteolyticdigestion between the two variants, and may be an indication ofdifferent biological activities or of different biological roles for thetwo variants. Such differences in stability and other biologicalproperties can possibly be turned to account in the use of thesehirudins.

The difference in the expression of HV1 and HV2 in E. coli can also beobserved by pulse-labeling analysis of E. coli TGE900 cells transformedby two different expression vectors. Since the two hirudin variants arevery rich in cysteine (approximately 10% of the molecule), and sincethis amino acid is rather uncommon in E. coli proteins, ³⁵ S!cysteine isvery useful as a radioactive marker for the expression of hirudin. E.coli cells transformed by pTG726 are grown at 30° C. to an opticaldensity of 0.3 determined in LB+ampicillin (100 μg/ml), and then inducedto express the variant HV1 by increasing the temperature to 37° C.

At regular hourly intervals, a 200 μl aliquot of the culture iswithdrawn and 70 μCi of ³⁵ S!cysteine (specific activity 1000 Ci/mmol)are added for a labeling period of 2 minutes. A large excess,approximately 2 ml, of cold phosphate buffered saline is then added, thecells are collected by centrifugation and the labeled proteins in thewhole cells are analyzed by boiling the pellet for 5 minutes in 40 μl ofa loading buffer for an SDS gel (50 mM Tris HCl, pH 6.8, 1.3% SDS, 5%glycerol, 2.5% β-mercaptoethanol, 0.004% bromophenol blue) and charge(sic) of 5 μl on a 15% SDS-polyacrylamide gel (procedure oflaemli-reference 36). After electrophoresis, the gel is subjected tofluorography followed by auto-radiography.

The results show the induction of a series of bands in the region of6,000 to 12,000 daltons, corresponding to hirudin. These bands are onlyweakly labeled with the E. coli/pTG726 extracts (variant HV1), whereasthey are very strongly labeled with the E. coli/pTG720 extracts.

This very distinct difference in the pattern of labeling appears inspite of the fact that the two cultures show approximately the samelevel of antithrombin activity.

Other properties of the hirudin recombinant HV1

One of the characteristics of natural hirudin, and also of the variantHV2 prepared by E. coli, is its resistance to heat treatment underconditions of quite low pH. This is also true for the variant HV1prepared from E. coli. A culture, induced for 3 hours, of E. coli cellstransformed by pTG726 is collected by centrifugation and resuspended in1/5 of the culture volume of TGE (50 mM Tris HCl, pH 8.0, 50 mM glucose,10 mM EDTA) and the cells are shattered by sonication. The cell debrisis removed by centrifugation and a portion of the supernatant is useddirectly for determination of the antithrombin activity.

Another portion, is adjusted to pH 2.8 with dilute HCl, and then heatedto 70° C. for 15 minutes. The mixture is then cooled in ice for 30minutes and the denatured insoluble proteins are removed bycentrifugation. The supernatant is neutralized by adding dilute NaOH andthe antithrombin activity is then measured. After the smallmodifications in volume due to the acidification and neutralization aretaken into account, it can be calculated that 100% of the originalactivity survives this acid/heat treatment. For this reason, the variantHV1 is identical to natural hirudin and to the variant HV2.

The hirudin HV2 activity can also be completely removed from a bacterialextract by using thrombin linked covalently to Sepharose beads. An E.coli/pTG726 extract treated with acid and heat and then neutralized, andcontaining 7.7 units of antithrombin activity in 200 μl, is incubatedwith 50 μl of a 50% strength suspension of thrombin-Sepharose for 15minutes at 37° C. The beads of thrombin-Sepharose are removed bycentrifugation and the supernatant is tested for its antithrombinactivity. More than 95% of the original antithrombin activity is removedby Sepharose-thrombin treatment. The variant HV1 produced by the E. colicells is consequently capable of binding to thrombin bound to Sepharosebeads.

The following strains were filed on Mar. 26th 1985 in the CollectionNationale de Cultures de Microorganisms (CNCM) (National Collection ofMicroorganism Cultures)-28 rue du Docteur-Roux-75724 PARIS CEDEX 15:

E. Coli TGE900 transformed by pTG718 No. I-427

E. Coli TGE900 transformed by pTG720 No. I-428

E. Coli TGE900 transformed by pTG726 No. I-429

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What is claim is:
 1. A vector for producing a hirudin variant in atransformed host procaryotic cell, said vector comprising a DNA fragmentencoding a hirudin variant having an amino acid sequence as follows:

    ______________________________________                                        Ile  Thr    Tyr    Thr  Asp  Cys  Thr  Glu  Ser  Gly                          Gln  Asn    Leu    Cys  Leu  Cys  Glu  Gly  Ser  Asn                          Val  Cys    Gly    Lys  Gly  Asn  Lys  Cys  Ile  Leu                          Gly  Ser    Asn    Gly  Lys  Gly  Asn  Gln  Cys  Val                          Thr  Gly    Glu    Gly  Thr  Pro  Asn  Pro  Glu  Ser                          His  Asn    Asn    Gly  Asp  Phe  Glu  Glu  Ile  Pro                          Glu  Glu    Tyr    Leu  Gln,                                                  ______________________________________                                    

said vector being capable of expressing said variant.
 2. A vectoraccording to claim 1, having an origin of replication which is ofbacterial origin.
 3. A vector according to claim 1, which is selectedfrom the group consisting of pTG720, pTG718 and pTG719.
 4. A procaryoticcell transformed with a vector according to claim
 1. 5. A cell accordingto claim 4, which is a bacterium.
 6. A cell according to claim 5, whichis of the E. coli species.
 7. A process for preparing a hirudin varianthaving an amino acid sequence starting at the N-terminal end with an Ileresidue immediately followed by a Thr residue, which comprises the stepsof culturing a procaryotic cell according to claim 4 and recovering fromthe culture said hirudin variant.
 8. A vector according to claim 1,which is pTG726.